c-Met and Epidermal Growth Factor Receptor (EGFR) are receptor tyrosine kinases (RTKs) that work together to promote the progression of Non-Small Cell Lung Cancer (NSCLC). Our current studies focus on the use of VeraTag technology to quantitatively, accurately and objectively determine if co- activation, co-overexpression and proximity of c-Met and EGFR can predict patient prognosis. Research over the last decade has enabled the development of several RTK-targeted inhibitors (RTKIs) against both c-Met and EGFR. However, recent clinical trials, targeting individual receptors or combinations, are not always effective due to their acquired resistance. Hence, it is important to determine the mechanism of resistance to EGFR/c-Met RTKIs, which is not defined, by utilizing drug resistant cell lines, developed in our lab. Future studies must focus on the development of new inhibitor combinations which may be targeted together to reduce resistance to EGFR/c-Met based therapies. The specific aims of this project are: Specific Aim 1: Determine the role of EGFR and c-Met expression in lung cancer tumorigenicity and elucidate the role of c-Met/EGFR mediated synergism and resistance in human tumor tissues. A. Determine the co-activation, co-overexpression and proximity of c-Met and EGFR in human lung tumor tissues through VeraTag technology and immunostaining for EGFR and c-Met phosphorylated forms and their ligands. Specific Aim 2: Elucidate the mechanism of c-Met and EGFR resistance. A. Determine if resistance developed to EGFR/c-Met RTKIs alters response to EGF or HGF and increases c- Met stability. B. Identify key downstream signaling proteins associated with acquired resistance to c-Met/EGFR therapies by western blotting/mass spectrometry and validate the efficacy of these proteins using si/shRNA vectors. C. Identify activated kinases mediating resistance to EGFR/c-Met inhibitors using kinase enrichment kits and active site probes. Study Design: To understand how c-Met and EGFR function together, we propose to study EGFR and c-Met co-overexpression, co-activation and proximity in NSCLC tumors using novel VeraTag technology and immunostaining. To study the mechanism of resistance to EGFR and c-Met RTKIs, cell lines resistant to EGFR, c-MET, and EGFR/c-Met combined RTKIs have been developed. Mass spectrometry and microarray analysis will be performed to identify specific proteins that may play an important role in resistance to c-Met and EGFR inhibitors. Using siRNA/shRNA vectors the role of a few novel target proteins that play a role in resistance to RTKIs will be validated. Kinase enrichment kits and active site probes will be used in collaboration with Thermo Fisher Scientific to study kinases which are activated specifically in resistant cells.